The B-cell lymphoma 2 (BCL-2) protein family plays a pivotal role in regulating the apoptosis process. BCL-2, as an antiapoptotic protein in this family, mediates apoptosis resistance and is an ideal target for cell death strategies in cancer therapy. Traditional treatment modalities target BCL-2 by occupying the hydrophobic pocket formed by BCL-2 homology (BH) domains 1–3, while in recent years, the BH4 domain of BCL-2 has also been considered an attractive novel target. Herein, we describe the discovery and identification of DC-B01, a novel BCL-2 inhibitor targeting the BH4 domain, through virtual screening combined with biophysical and biochemical methods. Our results from surface plasmon resonance and cellular thermal shift assay confirmed that the BH4 domain is responsible for the interaction between BCL-2 and DC-B01. As evidenced by further cell-based experiments, DC-B01 induced cell killing in a BCL-2-dependent manner and triggered apoptosis via the mitochondria-mediated pathway. DC-B01 disrupted the BCL-2/c-Myc interaction and consequently suppressed the transcriptional activity of c-Myc. Moreover, DC-B01 inhibited tumor growth in vivo in a BCL‑2‑dependent manner. Collectively, these results indicate that DC-B01 is a promising BCL-2 BH4 domain inhibitor with the potential for further development.

B 细胞淋巴瘤 2 (BCL-2) 蛋白家族在调节细胞凋亡过程中起着关键作用。BCL-2 作为该家族中的抗细胞凋亡蛋白，介导细胞凋亡抗性，是癌症治疗中细胞死亡策略的理想靶点。传统治疗方式通过占据由 BCL-2 同源 (BH) 结构域 1-3 形成的疏水口袋来靶向 BCL-2，而近年来，BCL-2 的 BH4 结构域也被认为是一个有吸引力的新靶点。在此，我们描述了通过虚拟筛选结合生物物理和生化方法发现和鉴定 DC-B01，一种靶向 BH4 结构域的新型 BCL-2 抑制剂。我们的表面等离子共振和细胞热位移分析结果证实，BH4 结构域负责 BCL-2 和 DC-B01 之间的相互作用。进一步基于细胞的实验证明，DC-B01 以 BCL-2 依赖性方式诱导细胞杀伤，并通过线粒体介导的途径触发细胞凋亡。DC-B01 破坏了 BCL-2/c-Myc 相互作用，从而抑制了 c-Myc 的转录活性。此外，DC-B01 以 BCL-2 依赖性方式抑制体内肿瘤生长。总的来说，这些结果表明 DC-B01 是一种很有前途的 BCL-2 BH4 结构域抑制剂，具有进一步开发的潜力。